We observed that E. coli engineered with proteorhodopsin produced more ATP when exposed to light, due to the activation of the proton pump (see Proteorhdopsin). We wanted to see if this makes bacteria more MFC friendly (i.e. live happily in the anode chamber) and if that would produce more electricity.

Cells transformed with BBa_K1604010 (araC-pBAD + proteorhodopsin) and BBa_K731201 (negative control) were grown in M9 media and induced with arabinose (5 mM) and all-trans retinal (10 μM) for 4 hours in darkness. Preliminary tests showed that the optimal medium to be used was M9 medium supplemented with glucose, which gave a more stable signal (data not shown).

The bacterial cultures were split and then placed in the anodic chamber of a small Microbial Fuel Cell (borrowed from one of our instructor Martin Hanczyc) and exposed to the light of a blue LED. The experiment was repeated for 3 days, keeping the same experimental conditions. In the presence of a blue-light LED, the proteorhodopsin expressing strain showed in all three cases a better electrochemical response than the negative control (i.e. PR-expressing strain shows higher polarization and power curves), with a higher voltage and maximum power (P_max). In a biological scale this means that there is an increased electricity due to the ability of the bacteria to maintain an active metabolism also in the absence of oxygen.^[1]

Small Microbial Fuel Cell with bacteria expressing BBa_K1604010 and BBa_K731201 in the light. Bacteria were placed in the anode covered with a layer of mineral oil to keep anaerobic conditions. The anode was exposed to blue light LED. Chemical mediators were added in the anode (Methylene blue, 100 μM) and in the cathode (Ferricyanide, 10 mM)

More electricity with proteorhodopsin! BBa_K1604010 and BBa_K731201 cells were grown and induced as described before. For each construct one MFC was placed in the light. The cells were connected to a data logging multimeter connected to an external variable resistor to register the voltage parameter of our system. Every hour the resistance was changed starting from 10MΩ to 1 KΩ Panel A: Polarization curve for BBa_K1604010 and BBa_K731201; for each data point the voltage was measured, while current and power were calculated with the Ohm law. Panel B: Power curve for BBa_K1604010 and BBa_K731201. The calculated power is plotted against the current to estimate the maximum power produced.

We wanted to investigate the ability of proteorhodopsin to respond to light. The cells were grown and induced as before. The same sample of cells expressing proteorhodopsin was divided post-induction in two equal samples. One sample was placed in the dark and one in the light. When exposed to light BBa_K1604010 showed a remarkable response to the external load applied, as shown by the higher values of voltage and current in the light. However, it has to be pointed out that this behavior was not always consistent. A few times we also observed the reverse effect (more electricity in the dark). This data are in agreement with the functional characterization, in which it was shown that a few times there was a basal activation of the proton pump also in the dark.

BBa_K1604010 polarization curve: light versus dark.The experiment was performed with the same experimental details described before. This time MFC with BBa_K1604010 was placed in the dark and one was exposed to the light of a blue LED.

All previous tests were operated by adding exogenous mediators to the anodic medium (i.e. Methylene blue, Neutral red). However this does not represent a valid method for future applications of the MFC. Related to our main project, we also characterized a mediatorless MFC by expressing Shewanella oneidensis electron export system in an engineered E.coli strain from Ajo-Franklin Lab in Berkeley ^[2]). We characterized this strain in the MFC because we wanted to use it later as a model bacteria in our MFC prototype. It should be noted that the parts used here were not BioBricks.

E.coli Mtr electron transport system polarization and power curve.C43(DE3) cotransformed with a IPTG inducible plasmid carrying the cymAmtrCAB operon and a plasmid with ccmA-H under pTet constitutive promoter, were grown in LB and induced with IPTG (0.5 mM). The induced cells were placed in a MFC without mediators. The data were acquired as described earlier for two biological sample of each part.

The increased electron flow we saw this time was mediated by the expression of Shewanella electron export complex. Such increase is not related to bacteria’s viability.

Last, we wanted to test pncb (BBa_K1604031), our own new device for the production of NAD+, to evaluate any possible improvement in electrons flow. Our characterization data for this part showed an increase in NAD+ intracellular concentration of 13 fold in anaerobiosis. When placed in a MFC the bacteria expressing pncB showed higher values of voltage and current for each resistance applied (data not shown) respect to the negative control. However, this was a preliminary result that we had no time to repeat do to the lack of time.

Polarization curve for BBa_K1604031 and BBa_K731201 Bacteria were placed in the anode of a small MFC covered with a layer of mineral oil to keep anaerobic conditions. Chemical mediators were added in the anode (Neutral red, 100 μM) and in the cathode (Ferricyanide, 10 mM). Voltage was measured with an external multimeter changing the external resistance. Current and power were calculated with the Ohm law.

Proteorhodopsin can power the blue-light LED used by UniTN iGEM Trento 2013 to produce ethylene! We used small MFCs filled with proteorhodopsin-expressing bacteria (BBa_K1604010), connected in series, to light up a few electronic apparatus, including a calculator, a blue-light LED and a lab timer.

Watch this video to see our MFC in action: